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Laboratorium voor Immunologie
Vakgroep Virologie, Parasitologie en Immunologie
Faculteit Diergeneeskunde
Universiteit Gent
Insights into the epidemiology of enteropathogens of
young pigs raised in Cuban piggeries
Pedro Yoelvys de la Fé Rodríguez
Promotor:
Prof. Dr. Bruno Maria Goddeeris
Co-promotoren:
Prof. Dr. Eric Cox en Prof. Dr. Luis O. Maroto Martin
Proefschrift voorgelegd aan de Faculteit Diergeneeskunde tot het behalen van de
graad van Doctor in de Diergeneeskundige Wetenschappen – Universiteit Gent, 2012
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Table of contents
List of abbreviations 7
Introduction 9
Chapter 1 General introduction 13
Aims of the study 45
Chapter 2 Association of toxigenic E. coli and several other pathogens
with porcine pre- and post-weaning diarrhea in Villa Clara
province, Cuba
2.1 Abstract 49
2.2 Introduction 50
2.3 Materials and methods 50
2.4 Results 54
2.5 Discussion 57
2.6 Acknowledgments 60
Chapter 3 Antibiotic resistance and genetic relatedness among
pathogenic E. coli isolated from intestinal contents of diarrheic
piglets in Villa Clara province, Cuba
3.1 Abstract 63
3.2 Introduction 64
3.3 Materials and methods 64
3.4 Results 66
3.5 Discussion 68
3.6 Acknowledgments 70
Chapter 4 High prevalence of F4+ and F18
+ E. coli in Cuban piggeries as
determined by serological survey
4.1 Abstract 73
4.2 Introduction 74
4.3 Materials and methods 74
4.4 Results 78
4.5 Discussion 86
4.6 Acknowledgments 88
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Chapter 5 Screening commercial pigs in Villa Clara province, Cuba, for
mucin 4 polymorphisms and susceptibility/resistance to F18+ E.
coli
5.1 Abstract 91
5.2 Introduction 92
5.3 Materials and methods 92
5.4 Results and Discussion 94
5.5 Acknowledgments 97
Chapter 6 General discussion and future perspectives
6.1 General discussion 101
6.2 Future perspectives 105
Summary 107
Samenvatting 111
Resumen 115
References 119
Curriculum vitae 143
Word of thanks 147
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List of abbreviations
7
List of abbreviations
ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
AIDA-I adhesin involved in diffuse adherence
B. coli Balantidium coli
C. parvum Cryptosporidium parvum
C. perfringens Clostridium perfringens
CPE C. perfringens enterotoxin
DAS-ELISA double-antibody-sandwich enzyme-linked immunosorbent assay
E. coli Escherichia coli
EAST-I enteroaggregative E. coli heat stable enterotoxin
EDTA
Ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
EM electron microscopy
EPEC enteropathogenic E. coli
ETEC enterotoxigenic E. coli
FAT fluorescent antibody technique
F18R F18 receptor
F4R F4 receptor
I. suis Isospora suis
IC immunochromatography
IF immunofluorescence
IFAT indirect fluorescence antibody technique
IHC immunohistochemistry
Int intimin
ISH In-situ hybridization
kDa kilo Dalton
LT heat-labile enterotoxin
MAb monoclonal antibody
OD optical density
PAA porcine attaching and effacing-associated factor
PAGE polyacrylamide gel electrophoresis
pAPN porcine aminopeptidase N
PCR polymerase chain reaction
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List of abbreviations
8
PCR-RFLP PCR-restriction fragment length polymorphism
PEDV porcine epidemic diarrhea virus
PRCV porcine respiratory coronavirus
PRRSV porcine respiratory and reproductive syndrome virus
RT-PCR reverse transcriptase PCR
ST heat-stable enterotoxin
TGE transmissible gastroenteritis
TGEV transmissible gastroenteritis virus
VN virus neutralization
VTEC verocytotoxigenic E. coli
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Introduction
9
Introduction
Porcine pre- and post-weaning diarrhea negatively impact the economic feasibility of the swine
industry due to mortality, costs of medication, and growth retardation (Francis, 1999; Katsuda et al.,
2006; Ushida et al., 2009). Housing and management conditions (e.g. hygiene, comfort temperature,
intake of maternal antibodies, feeding), vaccination against enteric pathogens, and surveillance of
antibiotic resistance are crucial aspects to consider during prevention of porcine diarrhea
(Fairbrother et al., 2005; Fairbrother, 2006; Straw et al., 2006).
Diarrhea of young pigs is frequently caused or complicated by rotavirus, transmissible
gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), enterotoxigenic Escherichia coli
(ETEC), toxigenic Clostridium perfringens, and Coccidia (Wieler et al., 2001; Straw et al., 2006;
Katsuda et al., 2006). Therefore, the differential identification of infectious agents is necessary to
evaluate diarrhea epidemiology in a swine herd (Collins et al., 1989; Niestrath et al., 2002; Nuñez et
al., 2003). However, reports of surveys aimed at studying the mixed condition of piglet’s infectious
diarrhea have been scarce worldwide (Wieler et al., 2001; Adesiyun et al., 2001; Yaeger et al., 2002;
Katsuda et al., 2006); many studies on porcine diarrhea have covered only single pathogens (Quilez
et al., 1996; Osek, 1999; Barreiros et al., 2003), and although in a TGEV/PEDV prevalence survey, also
pathogenic bacteria were identified, results were not shown (Chae et al., 2000). When performing
experiments related with the porcine digestive tract, the differential identification of
enteropathogens should be carried out as enteropathogens can significantly influence the results
(Jensen et al., 2006; Niestrath et al., 2010). Furthermore there is urgent need to better identify
synergisms or antagonisms among these pathogens (Baba and Gaafar, 1985; Choi et al., 2003).
ETEC are the most common cause of diarrhea in suckling and recently weaned pigs (Katsuda et
al., 2006). Cheng et al. (2006), Zhang et al. (2007), Madoroba et al. (2009), and Vidotto et al. (2009)
found that F4 or F18 fimbriae were the major fimbrial antigens expressed by pathogenic E. coli
associated with swine diarrhea. These fimbriae mediate the adhesion of ETEC to receptors present
on the surface of enterocytes, favoring gut colonization. The subsequent production of enterotoxins
by these bacteria leads to the secretion of electrolytes and water across the mucosa, resulting in a
watery diarrhea. Additionally, F18 favors the adhesion of verocytotoxigenic E. coli (VTEC) which
cause edema disease through a systemic vascular damage provoked by the verocytotoxin STx2e
(Fairbrother, 2006; Fairbrother and Gyles, 2006; Oanh et al., 2010).
Swine production is very important in Cuba as pork is the most consumed meat. In 2005
1,980,000 pigs were slaughtered and five years later already 3,266,600 according to the National
Office for Statistics (ONE, 2011).
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Introduction
10
In Cuban piggeries diarrhea is common and strongly reduces the survival rate of young pigs
leading to considerable economic losses: diarrheic diseases are responsible for 31% and 37% of the
total mortality during the pre- and post-weaning periods, respectively (Cabrera and García, 2009).
For instance, in the whole country 506,400 suckling piglets died in 2009 (12.2% mortality; ONE,
2011), and the 31% mortality provoked by diarrhea in this age group represent 156,984 deaths
(Cabrera and García, 2009).
It is contradictory that in Cuba specific and updated epidemiological information related to
swine enteropathogens is scarce. The prevention and control of diarrhea in Cuban piggeries are not
always well conducted due to lack of infrastructure in the Provincial Veterinary Diagnostic
Laboratories to properly perform the identification of enteropathogens (Cabrera et al., 2010).
Pedroso and Talavera (1983) showed in 1983 the presence of F4+ and F5
+ E. coli in feces of
piglets in the Havana province, whereas Blanco et al. (2006) could not isolate F4+, F5
+ or F41
+ E. coli
from diarrheic piglets in 2002 in the Villa Clara province. Most of their E. coli isolates (61%) were
F18+. The prevalence of Cryptosporidium parvum and Isospora suis was studied more than 20 years
ago in the Havana province in diarrheic piglets and was 2.1% and 44.7%, respectively (Cabrera and
García, 1985; Koudela et al., 1989). The first outbreak of epidemic transmissible gastroenteritis (TGE)
in Cuba was reported in Havana in 2003 (Barrera et al., 2005).
The present thesis contributes to the epidemiological characterization of enteropathogens
associated with porcine pre- and post-weaning diarrhea in Cuba, with special emphasis on
pathogenic E. coli. All epidemiological data obtained and discussed herein can be used for the
implementation of accurate preventive and therapeutic strategies to control porcine
enteropathogens in Cuba.
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Chapter 1
General introduction
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Chapter 1
13
Cuban context
In 1493, during his second trip to America, Christopher Columbus carried eight Iberian-trunk pigs to
Cuba on request of the queen Isabella I of Castile and León (Laguna, 1998). Nevertheless, for more than
four centuries during the colonial and neo-colonial periods, the production of cane sugar, alcohol,
tobacco, and leather were primordial in Cuba and pork was not an important production. By 1959, 68%
of swines were bred extensively. Approximately, 16% of the herds were intensively managed by
breeders and the remainder was a family-type production. From a genetic point of view, 22% of swine
were pure Criollo, and 69% were Criollo's crossbreds. Only 9% of swine herds had specialized breeds,
mainly Hampshire and Duroc. These genetic and management statuses did not allow an efficient swine
production leading to a high import dependency from the North American market before 1959 (Rico,
2005).
From the early 60's, as part of the changes that occurred in Cuba due to the 1959 Revolution, swine
production benefited from the following strategies:
i- Creation of the swine production department in the Ministry of Agriculture and elaboration of
a national policy for pig health and management.
ii- Foreign consultancy and staff training.
iii- Building of farms for intensive swine production and introduction of pure swine breeds into
breeding programs monitored by swine genetic centers located all over the country, which
ensured specialized swine production.
From 1971 to 1989, artificial insemination started to be applied and swine production was re-
organized under a pyramidal structure including genetic centers, multiplication piggeries (which
obtained replacement stock animals) and commercial piggeries. Also, swine sector benefited from
investments in facilities and from importations (mainly food and medicines) at preferential prices agreed
with the Council of Mutual Economic Assistance (COMECON). During this period, about 84% of swine
production occurred by the specialized state sector and the remaining 16% by private producers and
agricultural cooperatives (Rico, 2005).
From the 90's onwards, Cuban economy was negatively impacted by the dissolution of COMECON
due to the collapse of the communism in Eastern Europe and the Soviet Union, which were at that
moment the main trade partners and sources for technology transfer. Then, as happened with most
institutions, the services of the Cuban Veterinary Medicine Institute as well as swine production reduced
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Chapter 1
14
their activity. Specifically, the diagnostic effectiveness decreased and ongoing research on animal health
slackened, resulting in insufficient diagnosis of viral and bacterial agents. As a result, information
regarding swine enteric pathogens have been limited in Cuba:
· Pedroso and Talavera (1983) detected F4+ and F5
+ E. coli by immunofluorescence in feces of
piglets in the Havana province.
· The prevalence of C. parvum in diarrheic piglets from Havana was 2.5% in 1985 (Cabrera and
Garcia, 1985).
· 44.7% of diarrheic piglets were infected with I. suis in a piggery from Havana in 1989 (Koudela et
al., 1989).
· Fuentes et al. (2001) reported a 2.8% prevalence of F4+ E. coli in 212 diarrheic piglets sampled in
1998 in the Camagüey province.
· The first outbreak of TGEV was reported in 2003 in Havana and the disease was later
reproduced experimentally (Barrera et al., 2005).
· Blanco et al. (2006) genotyped E. coli isolates from diarrheic piglets in 2002 and found that most
of them (61%) carried the F18 encoding gene and that F4 encoding gene was absent.
The Cuban Institute for Swine Research recently reported that gastroenteric diseases cause 31%
and 37% of mortality in suckling and weaned piglets, respectively (Cabrera and García, 2009). In suckling
piglets, another causes of death, most of them closely related with diarrhea, such as crushing by the sow
(14%), malnutrition (8%), congenital diseases (e.g. myoclonia; 6%), low weight at birth (4%), and
hypoglycemia (3%) also require special attention. In weaned piglets, respiratory diseases (9%),
malnutrition (5%), and classic swine fever (5%) are common causes of death (GRUPOR, 2008; Cabrera
and García, 2009). Therefore, in 2008 the vice-director of the Cuban National Institute of Veterinary
Medicine recommended an improvement on swine management and on the specific diagnosis of enteric
diseases in order to decrease mortality by diarrhea in young pigs (Ricardo, 2008).
Nowadays, policies of the Cuban Ministry of Agriculture are to improve and increase swine
production as pork is the most consumed meat in the country. In 2008 the Cuban swine herd was
estimated to be 1,878,600 pigs (ONE, 2011), with the national company GRUPOR ruling the swine
production (Fig. 1; Rico, 2005).
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Chapter 1
15
Figure 1. Organization of the swine production in Cuba.
Table 1. Range of age at which the most common swine enteropathogens can cause disease.
Age of pigs
24 hours 5 days 3 weeks 5 weeks 10 weeks
Enteropathogens
TGEV
PEDV
Rotavirus
E. coli
Clostridium
Salmonella
Isospora suis
Strongyloides
National Institute of
Veterinary Medicine
Institute of Swine
Research
Provincial Swine
Companies
(GRUPOR)
National Company
for Pork Production
(GRUPOR)
Trading and Equipment
Company
State piggeries
(GRUPOR, Army,
Interior Ministry,
Ministry of Sugar)
Non-State farms
(Association of small
farmers, other
private farms)
Swine Genetic
Company (GRUPOR)
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Chapter 1
16
Multi-infectious etiology of diarrhea in young pigs
The clinical pictures of porcine pre- or post-weaning diarrhea often do not point to a specific etiology
because of variations in clinical signs and epidemiology that can be produced by the effect of one or
more disease agents. A control program of diarrhea could fail if one agent playing a primary role in the
pathogenesis remains sub-diagnosed. An explosive onset and rapid spread of diarrhea is usually
associated with a viral etiology. An insidious onset, slow spread, and gradual increase in severity over
time tend to be seen with bacterial or parasitic diseases (Straw et al., 2006).
A useful tool for veterinary practitioners regarding presumptive diagnosis of infectious diarrhea is the
age at which piglets are affected. But this is rather an indicator of which pathogens could be responsible,
and is not as precise as a specific etiologic identification test (Table 1; Straw et al., 2006).
The prevalence and repetitiveness of combined infections of enteropathogens reported worldwide
lead to the introduction of the term ¨mixed-type¨ during epidemiological analysis of diarrhea. However,
most prevalence studies on swine enteropathogens focused on single pathogens (Quilez et al., 1996;
Barreiros et al., 2003) and checking for a multi-infectious etiology has often been neglected (Chae et al.,
2000). Studies aimed at the differential identification of gastroenteric infections have displayed swine
diarrhea as a complex syndrome in which several pathogens can be associated (Table 3), explaining a
difficult control of pre- and post-weaning diarrhea in a piggery where enteropathogen identification has
not been well performed. A few surveys undertaken to differentially identify enteric pathogens in
suckling and weaned pigs with diarrhea indicate a high diversity of mixed infections which might lead to
or complicate diarrheal outbreaks in a piggery (Table 4). However, worldwide available data regarding
combined infections of enteropathogens remain difficult to compare as different tests were used for the
same pathogen or diagnoses did not cover all potential enteropathogens.
The main characteristics for differential diagnosis of swine gastroenteric infections were summarized
in Table 2 (adapted from Thomson, 2006).
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Chapter 1
17
Table 2. Insights for the differentiation of some common enteric diseases of young pigs.
Pathogens Age Signs Gross lesions Histological
lesions
Laboratory
methods
TGEV and PEDV All ages Watery diarrhea.
Rapid
dehydration.
Vomiting
Thin-walled pale
intestine, sparse
content
Severe villous
atrophy
PCR, IHC, ISH, or
IF of intestinal
contents.
Serology
Rotavirus 1 day to 7
weeks old.
Most
frequent at
2-3 weeks
Watery-pasty
diarrhea, sub-
clinical. Varying
dehydration
Fluid ingesta,
pale intestines.
Sparse stomach
contents
Moderate
villous atrophy
Virus detection:
PAGE, PCR,
ELISA, Tissue IHC
E. coli (ETEC,
EPEC)
Neonatal:
1-4 days.
Post-
weaning: 1-
3 weeks
after
weaning.
Watery, yellowish
diarrhea. Sudden
death.
Dehydration.
Fluid ingesta,
small intestinal
congestion,
watery content.
Stomach full of
milk.
Mucosal
congestion,
edema.
Bacterial
attachment to
gut epithelia.
Culture,
serotypes of
isolates, PCR.
Tissue IHC.
Agglutination
test.
C. perfringens
type C
1-14 days
(rarely
older)
Hemorrhagic
watery diarrhea.
Sudden death
Hemorrhagic
intestines,
mucosal
necrosis
Mucosal
necrosis,
associated
with Gram+
rods
C. perfringens
toxins ELISA’s on
intestinal
content.
Histopathology
Salmonella spp. All ages
after
weaning
Variable, watery
muco-
hemorrhagy. Most
infections
subclinical
Fibrinous or
hemorrhagic,
ulcers, intestinal
lesions
Ulcers,
neutrophil
infiltration,
fibrinous
thrombi
Culture,
serotyping,
phage type.
Antibody
detection.
Cryptosporidium
spp.
3 days to
weaning
Mild-moderate
yellowish diarrhea.
Varying degrees of
dehydration
Fluid ingesta None or mild
villous
atrophy.
Oocists in the
epithelium
Mucosal smear
for
cryptosporidial
oocysts.
Histopathology
I. suis 5-21 days Watery/yellowish
diarrhea.
Dehydration
Fluid ingesta,
necrosis of
intestinal
mucosa
Villous
atrophy,
fibronecrotic
enteritis,
intracellular
coccidians.
Stained mucosal
smear.
Histopathology.
Identification of
Coccidia
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Ch
ap
ter
1
18
Ta
ble
3.
Re
po
rts
on
dif
fere
nti
al
ide
nti
fica
tio
n a
nd
occ
urr
en
ce o
f e
nte
rop
ath
og
en
s in
su
ckli
ng
(S
) a
nd
we
an
ed
(W
) p
igs
suff
eri
ng
fro
m d
iarr
he
a w
orl
dw
ide
.
Pig
s P
ath
og
en
ic v
iru
ses
(%)
Pa
tho
ge
nic
ba
cte
ria
(%
)
Ro
tavir
us
Co
ron
avir
us
Sa
po
viru
s A
de
no
viru
s P
RR
SV
E
TE
C
Sa
lmo
ne
lla
C
. p
erf
rin
ge
ns
C.
dif
fici
le*
E
. d
ura
ns
S
8
59
.2 (
TG
EV
) N
T
0.5
3
NT
1
9.6
N
I 0
.40
(ty
pe
C)
NT
N
T
17
N
T
NT
N
T
NT
1
8.2
N
T
NT
N
T
NT
5.1
3
.4
NT
N
T
NT
7
.1
NI
NT
N
T
NT
42
6
(T
GE
V)
NT
N
T
15
9
**
N
I 6
NE
55
1
2
69
.3
0
5.3
N
T
NT
1
3.3
0
0
(ty
pe
C)
NT
N
T
20
.7
NT
N
T
NT
N
T
9.9
N
T
0 (
typ
e C
) N
T
NT
13
.3
NT
N
T
NT
N
T
10
N
T
43
.3 (
typ
e A
), 0
(C
) 2
3.3
N
T
W
0
51
.6
NT
N
T
NT
4
2.7
N
I N
T
NT
N
T
59
.3
0
16
.2
NT
N
T
54
.7
0
0 (
typ
e C
) N
T
NT
S&
W
4
13
.4
NT
N
T
NT
1
7.6
N
I N
T
NT
N
T
Pig
s P
ara
site
s (%
) N
eg
ati
ve
s (%
) C
ou
ntr
y
Re
fere
nce
I.
su
is
Eim
eri
a s
pp
. C
. p
arv
um
B
. co
li
He
lmin
ths
S
15
.3
0
NT
N
T
NT
9
.2
Ca
na
da
M
ori
n e
t a
l.,
19
83
53
.8
0
NT
N
T
NT
N
I A
ust
rali
a
Dri
ese
n e
t a
l.,
19
93
28
.8
NI
NI
NI
0
NI
Ge
rma
ny
W
iele
r e
t a
l.,
20
01
NT
N
T
NT
N
T
NT
N
I U
.S.A
. Y
ae
ge
r e
t a
l.,
20
02
18
.7 (
Co
ccid
ia)
0
NI
NI
17
Ja
pa
n
Ka
tsu
da
et
al.
, 2
00
6
18
.8 (
Co
ccid
ia)
NT
6
.9
NT
1
5.8
R
om
an
ia
Co
stin
ar
et
al.
, 2
01
0
0
NI
NI
NI
NI
NI
Bra
zil
Cru
z e
t a
l.,
20
10
W
1.2
N
I N
I N
I 0
N
I G
erm
an
y
Wie
ler
et
al.
, 2
00
1
18
.6 (
Co
ccid
ia)
16
.2
NI
NI
9.5
Ja
pa
n
Ka
tsu
da
et
al.
, 2
00
6
S&
W
20
.9
0.7
1
.4
0.7
0
5
7.7
G
erm
an
y
Wie
ler
et
al.
, 2
00
1
NT
, n
ot
test
ed
; N
I, n
o i
nfo
rma
tio
n;
*,
toxi
n d
ete
ctio
n;
**
, n
o d
ete
rmin
ati
on
of
vir
ule
nce
fa
cto
rs a
nd
se
roty
pe
s; N
E,
ne
cro
tic
en
teri
tis.
-
Chapter 1
19
Table 4. Occurrence (%) of enteric mixed infections in suckling (S)
and weaned (W) pigs with diarrhea reported worldwide.
¨Mixed types¨ Tested % S/W Country Reference
Rotavirus + PEDV 157 45.2
S South Korea Song et al., 2006
98 9.1 Czech Republic Czanderlova et al., 2010
Rotavirus + TGEV 749 0.8
S Canada Morin et al., 1983
98 4.5 Czech Republic Czanderlova et al., 2010
Rotavirus + TGEV + PEDV 98 22.7 S Czech Republic Czanderlova et al., 2010
Rotavirus + PRRSV 100 1 S U.S.A. Yaeger et al., 2002
Rotavirus + Sapovirus 153 1.3 S
Japan Katsuda et al., 2006 116 1.7 W
Rotavirus + Sapovirus + ETEC 153 2 S
116 2.6 W
Rotavirus + TGEV + ETEC 749 0.66 S Canada Morin et al., 1983
PRRSV + Clostridium difficile 100 3 S U.S.A. Yaeger et al., 2002
Rotavirus + Clostridium difficile 100 6
TGEV + ETEC 749 4.3 S Canada Morin et al., 1983
Rotavirus + ETEC
749 1.07
S
Canada Morin et al., 1983
1054 1.0 Australia Driesen et al., 1993
100 1.0 U.S.A. Yaeger et al., 2002
153 6.5 Japan Katsuda et al., 2006
116 19.0 W
Rotavirus + Sapovirus + ETEC +
Coccidia 116 6.0 W
Japan Katsuda et al., 2006
Rotavirus + Sapovirus + ETEC + C.
parvum 116 0.9 W
Sapovirus + ETEC + C. parvum 116 0.9 W
Rotavirus + Sapovirus + Coccidia 153 0.7 S
116 0.9 W
Rotavirus + Sapovirus + C. parvum 116 0.9 W
Rotavirus + TGEV + Coccidia 749 0.13 S Canada Morin et al., 1983
Rotavirus + ETEC + Coccidia
1054 0.6
S
Australia Driesen et al., 1993
101 2.9 Romania Costinar et al., 2010
153 1.3 Japan Katsuda et al., 2006
116 3.4 W
Rotavirus + ETEC + Coccidia + C.
parvum 116 2.6 W Japan Katsuda et al., 2006
Rotavirus + ETEC + Coccidia + B.
coli 101 0.9 S Romania Costinar et al., 2010
Rotavirus + Coccidia
9* 55.6
S
U.S.A. Roberts and Walker, 1982
749 1.2 Canada Morin et al., 1983
1054 6.7 Australia Driesen et al., 1993
101 8.9 Romania Costinar et al., 2010
153 7.2 Japan Katsuda et al., 2006
116 2.6 W
Rotavirus + Coccidia + B. coli 101 6.9 S Romania Costinar et al., 2010
Rotavirus + C. parvum 116 2.6 W Japan Katsuda et al., 2006
Sapovirus + Coccidia 153 0.7 S
TGEV + Coccidia 749 2.8 S Canada Morin et al., 1983
ETEC + Clostridium difficile 100 1.0 S U.S.A. Yaeger et al., 2002
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Chapter 1
20
Table 4 (continuation). Occurrence (%) of enteric mixed infections in suckling (S)
and weaned (W) pigs with diarrhea reported worldwide.
¨Mixed types¨ Tested % S/W Country Reference
ETEC + Coccidia
749 0.8
S
Canada Morin et al., 1983
1054 10.7 Australia Driesen et al., 1993
153 2.6 Japan Katsuda et al., 2006
116 0.9 W
ETEC + C. parvum 116 2.6 W Japan Katsuda et al., 2006
Occurrence of multiple infections
749 11.7
S
Canada Morin et al., 1983
1054 19.1 Australia Driesen et al., 1993
100 12 U.S.A. Yaeger et al., 2002
101 27.7 Romania Costinar et al., 2010
153 22.2 Japan Katsuda et al., 2006
116 47.4 W
92 9.0 Hungary Nagy et al., 1996
287 9.1 S&W Germany Wieler et al., 2001
In this table and in references to this table Coccidia refers to I. suis and or Eimeria spp.
As shown in Table 4, combined infections of Rotavirus-ETEC, Rotavirus-Coccidia, ETEC-Coccidia,
and Rotavirus-ETEC-Coccidia have been commonly reported. Despite a low occurrence, the
associations Rotavirus-ETEC-Coccidia-C. parvum, TGEV-Rotavirus-ETEC, and TGEV-Rotavirus-Coccidia,
are important to consider because of the proven pathogenic effect of the involved agents.
Katsuda et al. (2006) in Japan and Costinar et al. (2010) in Romania reported that 22.2% and
27.7% of suckling diarrheic piglets, respectively, carried mixed infections. Therefore, mixed
infections have to be accurately identified during diarrhea outbreaks to plan and implement an
efficient disease surveillance, prevention and control in every piggery, geographic area, or
management system.
Knowledge on the role of individual enteropathogens during pathogenesis and course of
combined enteric infections is limited. Practically, no controlled experiments have been designed to
study the interaction between the diverse enteropathogens; however, interesting findings have
been reported and discussed by some authors:
· Baba and Gaafar (1985) demonstrated that piglets which have been infected with S.
typhimurium and subsequently with I. suis, showed significantly smaller (p
-
Chapter 1
21
· Lecce et al. (1982) suspected that rotavirus provokes a damage of the epithelium that can
alter binding sites on enterocytes, favoring gut colonization by ETEC. Similar observations
were made by Melin et al. (2004).
· Necropsy seventeen days post-Ascaris infection revealed lower hepatic fibrosis and a lesser
degree of liver eosinophilia in Ascaris infected pigs pre-inoculated with Salmonella than
those receiving Ascaris inoculation only (Wade and Gaafar, 1981). Similar results were seen
in pigs recovered from TGE and later infected with Ascaris (Gaafar et al., 1973).
· Janke et al. (1988) concluded that rotaviral infection in colostrum-deprived piglets is
inhibited by enteroviruses infection.
· Choi et al. (2003), after using plant lectins for studying the histochemistry of the jejunal
mucosa of pigs infected with I. suis, argue that variations on the glycoconjugates
composition due to isosporosis could favor E. coli colonization.
· Enemark et al. (2003a) found that mixed infections of rotavirus and Cryptosporidium cause a
dramatic aggravation of diarrhea and clinical signs; piglets mono-infected with Rotavirus or
Cryptosporidium showed no or very mild clinical signs of illness.
· C. perfringens type C may colonize lesions initiated by Coccidia, rotavirus, TGEV and PEDV
(Songer and Uzal, 2005).
· Zintl et al. (2007) found that Cryptosporidium infections are not commonly combined with
Salmonella infections in pigs.
· Jung et al. (2008) proposed that the severe diarrhea in PEDV-rotavirus A co-infected piglets
may be more associated with the immunity level of the host rather than to any synergistic
effect of rotavirus on PEDV enteritis. They suggested that concurrent infection with porcine
rotavirus A does not synergistically enhance intestinal villous atrophy.
· Kim et al. (2010a) reported that 124/182 (68%) rotavirus A infections were mixed with other
enteric pathogens.
Major enteropathogens causing diarrhea in young pigs
Escherichia coli
E. coli are an important cause of diarrhea in suckling and recently weaned pigs, and are
responsible for significant economical losses worldwide. Most porcine ETEC and VTEC strains
produce fimbriae, which enable them to colonize the epithelial surface of the porcine small
intestine. Pathology is the result of the production of toxins. ETEC strains produce heat-stable (STa
or STb) or heat-labile (LT) which induce fluid loss resulting in diarrhea (Nataro and Kaper, 1998).
-
Chapter 1
22
VTEC produce the verocytotoxin STx2e which is responsible for edema disease. Some E. coli strains
can produce both enterotoxins and the verocytotoxin STx2e which is responsible for edema disease,
and are appropriately referred to as ETEC/VTEC (Blanco et al., 2006).
Attachment to specific receptors is essential for colonization and pathogenesis. Fimbriae mediate
the attachment of E. coli to the intestinal mucosal surface. F4 and F18 fimbriae have been commonly
identified in E. coli associated with PWD. But, whereas F18 fimbriae are almost exclusively associated
with PWD, F4 fimbriae are also a predominant colonization factor implicated in neonatal diarrhea. Of
F4, 3 serotypes have been described, namely F4ab, F4ac and F4ad, of which F4ac is the most
prevalent (Choi and Chae, 1999). Of F18, two serotypes have been identified: F18ab and F18ac.
F18ab is more expressed by VTEC strains and F18ac by ETEC strains. Other fimbriae such as F5, F6,
and F41 are mainly expressed by porcine ETEC isolated from newborn piglets with diarrhea.
Nevertheless, F4 and to a lesser extent F18, continue to be the major fimbrial antigen types in ETEC
and VTEC identified in diagnostic laboratories in the U.S.A. (Moon et al., 1999), China (Cheng et al.,
2006), Brazil (Vidotto et al., 2009), Zimbabwe (Madoroba et al., 2009), and Vietnam (Oanh et al.,
2010).
Some candidate receptors have been suggested for F4+ E. coli in the gut epithelium of pigs (F4R).
Erickson et al. (1992; 1994) proposed the intestinal mucin-like sialoglycoproteins (F4acR; IMTGPs),
Grange and Mouricout (1996) reported an intestinal transferrin (F4abR) and Grange et al. (1999) an
intestinal glycosphingolipid (F4adR). Recently, Rasschaert (2008) identified porcine aminopeptidase-
N (pAPN) as a receptor for F4ac. Looking at binding to glycosphingolipids, Coddens et al. (2011)
found that F4ab binds galactosylceramide, sulfatide, sulf-lactosylceramide and
globotriaosylceramide present in epithelial cells of the porcine intestine, whereas F4ac only binds
galactosylceramide.
Not all pigs have receptors for adhesion of F4+ and F18
+ E. coli. Some pigs are receptor negative.
Testing in vitro adhesion of the three F4 variants (ab, ac, and ad) to brush border membranes of
enterocytes, isolated enterocytes or isolated villi, six phenotypes could be identified: phenotype A
binds all three F4 variants, phenotype B F4ab and F4ac, phenotype C F4ab and F4ad, phenotype D
F4ad, phenotype E none of the variants, and phenotype F F4ab only. Based on that classification,
prevalence studies have been done in different swine herds: in Midwestern United States and The
Netherlands 30% and 50% of pigs showed the phenotype E, respectively (Sellwood et al., 1975,
Baker et al., 1997; Bijlsma et al., 1985). Conversely, 80% of the Belgian pigs were classified in the F4
susceptible phenotype A, and only 4% in the resistant phenotype E (Cox and Houvenaghel, 1987).
The F4ab/ac receptor loci are closely linked and linked to the transferrin locus on chromosome
13. They behave as a single autosomal dominant gene (Guérin et al., 1993). Python et al. (2002)
-
Chapter 1
23
concluded that the receptor for F4ac binds F4ab bacteria as well, and that it is controlled by one
gene localized between S0068 and Sw1030 on chromosome 13. Jørgensen et al. (2004) detected
that a mutation G→C in intron 7 of mucin 4 gene was strongly associated with the ETEC F4ab/ac
adhesive phenotype. They developed a PCR-RFLP which detected polymorphisms in a mucin 4 gene
fragment by its digestion with XbaI enzyme, and allows genotyping pigs for resistance or
susceptibility to adhesion of E. coli mediated by F4ab/ac fimbriae. Three different pig genotypes can
be observed: resistant (no adhesion) homozygote carrying indigestible alleles (RR) and susceptible
heterozygote (SR) as well as homozygote (SS). Conversely, Rasschaert et al. (2007) could not confirm
this good correlation when comparing results of the DNA-based marker test with results of the in
vitro villous adhesion assay. A reason for the difference in both studies could be the use of a
different in vitro adhesion assay as Jørgensen et al. (2004) used single enterocytes whereas
Rasschaert et al. (2007) used isolated villi.
Looking at binding of F18 to glycosphingolipids, Coddens et al. (2009) could identify the receptors
for F18. They observed a high specific interaction of F18 E. coli with glycosphingolipids having blood
group A/B/H determinants on type 1 core chains, as well as the blood group A type 4
heptaglycosylceramide.
The in vitro villous adhesion tests also allow discrimination between F18R+ and F18R
- pigs.
Absence or presence of the F18R is genetically determined and susceptibility being dominant over
resistance (Bertschinger et al., 1993). The gene controlling expression of the F18R was mapped to
the halothane linkage group on pig chromosome 6 (Meijerink et al., 1997). This locus contained two
candidate genes, FUT1 and FUT2, both encoding α2-fucosyltransferases. Expression analysis of these
two genes in the porcine small intestine revealed that only the FUT1 gene was expressed in all
examined pigs (Meijerink et al., 2000). This gene is localized in chromosome 6q11 and the enzyme
alpha (1,2)-fucosyltransferase 1 transfers fucose to lipid, carbohydrate, and protein backbones
present in the intestine. Sequencing of this gene showed a polymorphism (G or A) at nucleotide 307
resulting in an amino acid substitution at position 103 (Ala/Thr) of the enzyme. Presence of the A
nucleotide on both alleles (FUT1A/A genotype) led to significantly reduced activity of the enzyme
corresponding to the F18-fimbriated E. coli resistant genotype, whereas susceptible pigs had either
the heterozygous FUT1G/A or the homozygous FUT1G/G genotype. These findings have led to the
development of a PCR-RFLP using CfoI enzyme that allows the classification of pigs into susceptible
or resistant to F18+ E. coli adhesion (Meijerink et al., 1997).
Frydendahl et al. (2003) found a high correlation between F18R+ genotypes and susceptibility to
F18+
E. coli; however, pigs carrying the resistant F18R genotype were not entirely protected against
intestinal colonization. Coddens et al. (2007) also found a significant positive but weak correlation
-
Chapter 1
24
(r=0.307, p
-
Chapter 1
25
resistance were found to be co-located on a self-conjugative plasmid (pTC, 120-kb) which is widely
distributed among porcine ETEC (Olasz et al., 2005).
As good management, hygiene and vaccination in swine farms, also antibiotics have helped in
preventing and controlling swine colibacillosis, but it also provoked the appearance and selection of
resistant and multidrug-resistant E. coli which tend to persist in time and space (Maidhof et al.,
2002; Blake et al., 2003; Moredo et al. 2007; Dewulf et al., 2007; Akwar et al. 2008; Vieira et al.,
2009; Bibbal et al., 2009). In New Zealand, Nulsen et al. (2008) found a higher antibiotic resistance to
tetracycline (60% v/s 5%), streptomycin (25% v/s 3%), and cotrimoxazole (11% v/s 0%) in E. coli
isolated on conventional farms (where is a higher antibiotic pressure) than on organic farms.
Maynard et al. (2003) concluded that the genes behind phenotypic antibiotic resistance are not
static and their prevalence is determined by various selection forces such as the use of specific
antimicrobials.
Investigation of virulence factors, antimicrobial resistance, and genetic profiles are essential to
deeply study the epidemiology of ETEC associated with swine diarrhea. ETEC carrying the same
virulence profiles and with similar antibiogram sensitivity are likely to show a highly similar
pulsotype by PAGE (Lee et al., 2009; Bibbal et al., 2009). Such mixed studies are becoming an
important epidemiological tool. Thorsteinsdottir et al. (2010) found the same resistance profile and
pulsotype among E. coli isolated from broiler meat and slaughterhouse workers. They stated that
isolates sharing the same genetic profile and resistance patterns can arise on different farms.
Conversely, Rosengren et al. (2009) and Smith et al. (2010) did not find clear associations between
antimicrobial resistance and virulence profile in E. coli isolated from healthy versus diseased pigs.
Surveillance studies of swine colibacillosis should be applied to every geographic area or even at
farm or production system level as a high variety of antibiotics, resistance genes, virulence genes,
and their co-location onto conjugative plasmids or pathogenicity islands in E. coli lead to diverse
associations or clones (Hendriksen et al., 2008; Harada et al., 2008; Wang et al., 2010; Smith et al.,
2010). Additionally, the co-selection of E. coli resistant to some antibiotics (i.e. kanamycin) by the
use of other antibiotics (i.e. tetracycline) is contributing to increase antibiotic resistance in swine
farms. Therefore risk assessment has to be performed for every antibiotic or every chemical group in
order to better police the selection and persistence of resistant E. coli (Harada et al., 2008).
-
Ch
ap
ter
1
26
Ta
ble
5.
Re
cen
t st
ud
ies
rep
ort
ing
vir
ule
nce
en
cod
ing
ge
ne
s in
E.
coli
iso
late
d f
rom
fe
ces
of
yo
un
g p
igs
wo
rld
wid
e.
Sy
nd
rom
e
& a
ge
gro
up
n
% f
rom
te
ste
d i
sola
tes
Co
un
try
/Re
fere
nce
V
F+
F
4
F5
F
6
F4
1
F1
8
Int
ST
a
ST
b
LT
ST
x2e
E
AS
T1
ND
(<
21
d.)
2
00
6
3
33
.5
10
.5
0
0
0
NT
5
8
52
.5
35
N
T
NT
V
ietn
am
/Do
et
al.
, 2
00
6
(<
30
d.)
1
3
46
.2F, 1
00
T
0
0
23
0
2
3
NT
5
3.8
6
1.5
7
.7
7.7
N
T
Cu
ba
/Bla
nco
et
al.
, 2
00
6
(<1
4 d
.)
He
alt
hy
22
0
30
56
F,
74
T
17
F,
26
.7T
38
0
3
0
3
0
3
0
9
0
3
17
13
0
49
0
42
0
4
0
65
26
S
lova
kia
/Vu
-Kh
ac
et
al.
, 2
00
7
(4-2
1 d
.)
19
6
32
T
9.7
7
.7
0.5
7
.7
8.8
N
T
9.2
1
5.8
1
6.3
8
.8
NT
Z
imb
ab
we
/Ma
do
rob
a e
t a
l.,
20
09
ND
&P
WD
(0
-35
d.)
8
3
79
.3F,
90
T
45
.8
18
.4
26
.3
5.3
2
6.3
1
0.5
8
0.7
4
4.6
4
5.8
1
5.7
N
T
Jap
an
/Ka
tsu
da
et
al.
, 2
00
6
(2
-72
d.)
5
62
3
4
2.3
2
2
.5
2.8
5
.3
7.1
7
.3
4.8
7
.8
5.2
1
3.9
S
ou
th K
ore
a/K
im e
t a
l.,
20
10
b
PW
D
(4
-6 w
.)
He
alt
hy
37
2
46
29
F,
80
.1T
3.5
F,
8.7
T
19
.1A
0A
2.1
A
4.3
A
1.1
A
0A
2.9
A
0A
2.7
2.2
NT
NT
12
.4
2.2
4.3
0
57
.3
2.2
20
.4
4.3
NT
NT
P
ola
nd
/Ose
k,
19
99
(4-1
0 w
) 2
15
5
0.2
F,
7.2
T
9.8
A
10
.7A
15
.8A
9.8
A
25
.6A
NT
7
4.4
1
4
2.3
8
.8
NT
C
hin
a/C
he
n e
t a
l.,
20
04
(>3
0 d
.)
23
8
2.6
F,
10
0T
0
0
0
0
82
.6
NT
6
5.2
7
3.9
4
.3
78
.2
NT
C
ub
a/B
lan
co e
t a
l.,
20
06
-
10
1
60
F,
77
T
19
0
.9
5
0.9
3
5
0.9
2
6
46
2
0
5
64
S
lova
kia
/Vu
-Kh
ac
et
al.
, 2
00
6
- 3
04
5
8F
37
.1
0.3
0
0
.3
19
.7
0.7
D
15
.8
41
.8
33
.2
9.9
2
0
U.S
.A./
Zh
an
g e
t a
l.,
20
07
- 1
00
1
00
4
4
30
2
5
32
3
8
NT
4
0
47
7
1
3
NT
B
razi
l/V
ido
tto
et
al.
, 2
00
9
PW
D&
ED
- 2
30
4
0.9
1
0
1.7
4
.3
0.8
1
8.3
N
T
27
.5
15
.2
8.7
1
5.2
N
T
So
uth
Ko
rea
/Kw
on
et
al.
,
20
02
(4
-8 w
.)
21
9
85
.4F,
87
.2T
44
.7
0
0.9
0
3
9.3
1
.4
26
.5
7.6
6
1.6
1
6.4
6
5.8
D
en
ma
rk/F
ryd
en
da
hl,
20
02
- 2
40
1
00
3
.7
0
0
0
26
.2
28
.3
14
.5
9.1
1
0.8
3
5
NT
C
hin
a/C
he
ng
et
al.
, 2
00
6
ND
, n
eo
na
tal
dia
rrh
ea
; P
WD
, p
ost
-we
an
ing
dia
rrh
ea
; E
D,
ed
em
a d
ise
ase
; n
, te
ste
d E
. co
li i
sola
tes;
VF
, vi
rule
nce
fa
cto
r; T
an
d F
, p
osi
tive
fo
r a
t le
ast
on
e o
f th
e
test
ed
to
xin
or
ad
he
sin
ge
ne
s re
spe
ctiv
ely
; A,
ide
nti
fie
d b
y t
he
ag
glu
tin
ati
on
te
st;
D,
Zh
an
g e
t a
l. (
20
07
) a
lso
re
po
rte
d A
IDA
-I (
15
.5%
) a
nd
PA
A (
34
.5%
); N
T,
no
t
test
ed
; d
., d
ay
s o
ld;
w.,
we
ek
s o
ld.
-
Chapter 1
27
Clostridium perfringens
C. perfringens is a Gram-positive anaerobic bacterium that is able to form spores. It is widespread in
the environment (e.g. in soil and sewage) and is commonly found in the intestine of animals. C.
perfringens strains are classified into five toxinotypes (A, B, C, D, and E) according to the production of
alfa (α), beta (β), epsilon (ε) and or iota (ι) toxins which are crucial in the pathogenesis of clostridiosis
(Petit et al., 1999); besides, they can also produce a pore-forming enterotoxin called C. perfringens
enterotoxin (CPE; McClane, 1996) and a cytotoxic β2-toxin (Garmory et al., 2000) which are usually
tested for subtyping.
Alfa and β-toxigenic strains have been associated with swine diarrhea worldwide (Morin et al., 1983;
Niestrath et al., 2002; Yaeger et al., 2002; Das et al., 2009; Cruz et al., 2010).
C. perfringens type-C produce α and β toxins and cause hemorrhagic, often fatal, necrotic enteritis in
young piglets. The disease is most frequent in 3-day-old piglets, but it can appear in the first 12 hours of
life (Songer and Meer, 1996; Songer and Uzal, 2005). This strain is rarely found in the intestine of
healthy piglets. It is important to remark that piglet-piglet transmission occurs, and spores persist in the
environment as they are resistant to heat, disinfectants, and ultraviolet light. The main source of
infection in a piggery is the intestine of the sow (Songer, 1996). Type C clostridiosis can occur
epidemically in non-vaccinated herds, and prevalence in affected litters can reach 100% with mortality
close to 100% (Songer and Uzal, 2005).
The basis of the pathogenicity of C. perfringens type A strains (α-toxigenic) frequently isolated from
pigs with enteritis has not been clearly established but necrotic intestinal lesions have been
experimentally induced by inoculation of C. perfringens type A culture supernatant (Songer, 1996). The
enteropathogenicity of these strains might result from high levels of α-toxin production, from molecular
variants that are more stable to protease digestion or are more active, from different host sensitivity to
α-toxin (Ginter et al., 1996), or due to the side production of β2-toxin (Garmory et al., 2000; Hendriksen
et al., 2006) or the C. perfringens enterotoxin.
The α-toxin is a phospholipase C sphingomyelinase that hydrolyzes phospholipids (e.g. lecithin) and
promotes membrane disorganization, resulting in blood vessel contraction, increased vascular
permeability, platelet aggregation and myocardial dysfunction, all of which contribute to local and
systemic clinical manifestations (Bunting et al., 1997; Naylor et al., 1998). The β1- and β2-toxins induce
hemorrhagic necrosis of the intestinal mucosa (Gibert et al., 1997). Although these toxins are cytotoxic
(Gibert et al., 1997), their mode of action has not yet been completely elucidated. The fact that β1-toxin
displays a significant homology at the amino acid level with α-toxin, and leucocidin of Staphylococcus
-
Chapter 1
28
aureus which form multimers and pores in eukaryotic cell membranes, suggests that β1-toxin has a
similar mode of action (Hunter et al., 1993). Gurtner et al. (2010) confirmed that β1-toxin causes
disruption of the actin cytoskeleton of endothelial cells. Also, C. perfringens secretes a variety of
hydrolytic enzymes that degrade extracellular substrates and components resulting from cell lysis. It is
possible that these enzymes act synergistically with membrane-damaging toxins during cell disruption
(Petit et al., 1999). Zeng et al. (2011) developed and recommended the application of recombinant
fusion toxoids as good vaccine candidate against the α, β1, and β2 clostridial toxins.
Testing for C. perfringens toxins by enzyme-linked immunosorbent assay (ELISA) is a reliable test
(Naylor et al., 1997; Niestrath et al., 2002), i.e. the commercially available Bio-X ELISA kit (Bio-X
Diagnostics, Marche-en-Famenne, Belgium) that detects the α-, β- and ε-toxins in intestinal contents or
culture supernatants. Genotyping of C. perfringens has simplified the routine diagnosis. Multiplex-PCR
for detecting fragments of genes encoding toxins enables the diagnostician to screen larger numbers of
samples with higher accuracy and greatly reduces the amount of false-negative results (Das et al., 2009;
Baker et al., 2010).
In summary, the clinics and the differential diagnosis with other enteropathogens is very important
to consider when implicating C. perfringens as primary agent. Toxinotype A is widespread in the
intestines of pigs worldwide (Table 6) and its association with diarrhea has to be carefully analyzed in
every outbreak. Type C strains frequently cause hemorrhagic necrotic enteritis in young pigs (Petit et al.,
1999).
-
Ch
ap
ter
1
29
Ta
ble
6.
Re
po
rts
of
surv
ey
s a
sse
ssin
g a
sso
cia
tio
n o
f C
. p
erf
rin
ge
ns
wit
h s
win
e d
iarr
he
a w
orl
dw
ide
.
Ag
e
gro
up
s T
est
ed
%
A
sso
cia
ted
to
xin
s A
ssa
y
Co
un
try
/Re
fere
nce
α
β
1
β2
ε
C
PE
ι
1-1
5 d
. 7
49
0
.4
+N
E
+N
E
Cli
nic
s, h
isto
pa
tho
log
y g
en
era
l b
act
eri
olo
gic
pro
ced
ure
s
Ca
na
da
/Mo
rin
et
al.
,
19
83
1-3
d.
13
1
00
M
+
Ba
sed
on
an
am
ne
sis,
dif
fere
nti
al
dia
gn
ose
,
his
top
ath
olo
gy
, g
en
era
l b
act
eri
olo
gic
pro
ced
ure
s, i
no
cula
tio
n b
ioa
ssa
y a
nd
re
vers
e
pa
ssiv
e l
ate
x a
gg
luti
na
tio
n a
ssa
y
U.S
.A./
Co
llin
s e
t a
l.,
19
89
Su
cke
rs
33
8
7.9
+
-
+*
-
- -
Ge
ne
ral
ba
cte
rio
log
ic p
roce
du
res,
PC
R
U.S
.A./
Ga
rmo
ry e
t a
l.,
20
00
3
3
12
+
+
+
-
- -
7N
D
0
- -
- -
- -
Su
cke
rs
10
DN
50
I.s.
+
- N
T
- N
T
NT
D
AS
-ELI
SA
G
erm
an
y/N
iest
rath
et
al.
, 2
00
2
1-7
d.
10
0
6
+C
+C
Ne
cro
tizi
ng
in
test
ina
l le
sio
ns
in a
sso
cia
tio
n w
ith
larg
e,
gra
m-p
osi
tive
ba
cill
i li
nin
g n
ecr
oti
c vi
llu
s
rem
na
nts
. D
en
se g
row
of
C.
pe
rfri
ng
en
s
U.S
.A./
Ya
eg
er
et
al.
,
20
02
Su
cke
rs
22
0A
90
.9
+
- +
-
- -
PC
R (
du
rin
g t
his
su
rve
y C
.p.
iso
late
s w
ere
on
ly
test
ed
fo
r cp
b2
ge
ne
wh
ich
co
de
s fo
r β
2 t
oxi
n)
U.S
.A./
Bu
esc
he
l e
t a
l.,
20
03
3
6C
97
.2
+
+
+
- -
-
9C
.p.
ND
1
1.1
-
- +
-
- -
Su
cke
rs
15
3
0
- -
NT
-
NT
-
Ge
ne
ral
ba
cte
rio
log
ic p
roce
du
res,
PC
R
Jap
an
/Ka
tsu
da
et
al.
,
20
06
W
ea
ne
rs
11
6
0
- -
NT
-
NT
-
12
-14
m.
14
6
2.1
T
+
- +
-
- -
Ge
ne
ral
ba
cte
rio
log
ic p
roce
du
res,
PC
R
Ma
lay
sia
/Da
s e
t a
l.,
20
09
2
-3 m
. 9
6
2.1
T
+
- +
-
- -
1-7
d.
30
3
3.3
+
-
+
- -
- G
en
era
l b
act
eri
olo
gic
pro
ced
ure
s, P
CR
B
razi
l/C
ruz
et
al.
, 2
01
0
Su
cke
rs
33
3
89
.8
+
- N
T
- -
- G
en
era
l b
act
eri
olo
gic
pro
ced
ure
s, P
CR
U
.S.A
./B
ak
er
et
al.
, 2
01
0
NE,
ba
sed
on
fin
din
gs
of
ne
cro
tic
en
teri
tis
wh
ich
is
ass
oci
ate
d w
ith
C.
pe
rfri
ng
en
s C
; M
, m
orb
idit
y c
lose
to
10
0%
an
d 1
3 p
igle
ts (
8 s
ick
an
d 5
he
alt
hy
) w
ere
sele
cte
d f
or
C.
pe
rfri
ng
en
s st
ud
y;
*,
in 7
9%
of
C.
pe
rfri
ng
en
s A
; N
D, n
on
dia
rrh
eic
co
ntr
ol
pig
lets
, I.
s.,
mix
ed
wit
h I
. su
is,
an
d 1
/5 p
osi
tive
pig
lets
ha
d n
ecr
oti
c
en
teri
tis;
C,
C.
pe
rfri
ng
en
s ty
pe
C;
A,
C.
pe
rfri
ng
en
s ty
pe
A; C
.p.
ND,
C.
pe
rfri
ng
en
s is
ola
ted
fro
m n
on
dia
rrh
eic
pig
lets
; T,
C.
pe
rfri
ng
en
s w
as
iso
late
d f
rom
pig
s w
hic
h
die
d a
fte
r te
tra
cycl
ine
re
sist
an
t a
cute
dia
rrh
ea
; D
N,
dia
rrh
eic
an
d n
on
-dia
rrh
eic
pig
s.
-
Chapter 1
30
Transmissible gastroenteritis virus and porcine epidemic diarrhea virus
TGEV belongs to the Coronaviridae family. The virus contains a single-stranded genomic RNA and
only one serotype is known (Kemeny, 1976). Three major structural proteins described for coronaviruses
are the spike glycoprotein (S; 180–200 kDa), the membrane protein (M; 21–30 kDa), and the
nucleoprotein (N; 45–50 kDa) (Spaan et al., 1988). The S protein is the most interesting protein from an
antigenic and immunogenic point of view (Delmas et al., 1986; Jiménez et al., 1986; Torres et al., 1995).
Four antigenic sites (C, B, D, and A) were mapped on the S protein starting from the N-terminal end and
antibodies against them can be found in the serum of TGEV-infected pigs (Correa et al., 1990). The spike
glycoprotein initiates infection by binding to the enterocytes via pAPN, which has been identified as a
coronavirus receptor (Delmas et al., 1992; Hansen et al., 1998). PEDV, another coronavirus, also binds
specifically to pAPN and this binding can be inhibited by anti-pAPN antibodies (Oh et al., 2003; Li et al.,
2007). TGEV shows also sialic acid binding activity, perhaps providing a second binding site that may
account for the enteropathogenicity of the different strains (Schwegmann-Wessels et al., 2002;
Schwegmann-Wessels and Herrler, 2006).
Three swine coronaviruses are pathogenically or antigenically related: TGEV, PEDV, and porcine
respiratory coronavirus (PRCV). TGEV and PRCV cross-react serologically and are very closely related
only differing in a deletion mutation of 224-227 amino acids in the Spike protein S of PRCV in
comparison with TGEV. PRCV cannot be distinguished from enteropathogenic strains of TGEV by a virus
neutralization test (Callebaut et al., 1988). Indeed, infection with PRCV induces the production of
antibodies able to neutralize both TGEV and PRCV at the same titer (Pensaert et al., 1986). Previous
PRCV infections in piglets and or sows, or concurrent TGEV/PRCV infections influence and change the
pathogenesis of TGEV, thereby reducing the severity of disease (Cox et al., 1993; Kim et al., 2000). As a
consequence, the presence of PRCV in Europe reduced the incidence and severity of epidemic TGE
(Pensaert et al., 1993). Therefore, a low prevalence (0.9%) of TGEV infection compared with PEDV in
South Korea could be due to the high prevalence of PRCV (Chae et al., 2000). Also in the United States
and Japan, a decrease in TGE incidence has been reported in areas with a high prevalence of anti-PRCV
antibodies (Yaeger et al., 2002; Miyazaki et al., 2010). In TGEV- and PRCV-seronegative herds, however,
TGE remains a major cause of sickness and death in piglets (Barrera et al., 2005; Saif and Sestak, 2006).
Characteristics of TGE acute form are a short incubation period, diarrhea, vomiting, and dehydration.
Mortality approaches 100% in newborn piglets, but decreases with age. Sows infected shortly after
parturition may be severely affected by diarrhea, hypogalactia, and agalactia (Djurickovic et al., 1969;
Saif and Sestak, 2006). The first outbreaks of TGE reported in February 2003 in Cuba were a classic
-
Chapter 1
31
example of this form. On affected farms, 100% of recently farrowed sows and their litters had diarrhea.
The clinical signs in newborns included very liquid and fetid, yellowish feces and vomiting, leading to
serious dehydration. At the onset of the disease, sows showed a lack of appetite followed by vomiting
and agalactia, but all recovered. The weaned and fattening pigs of these farms presented severe clinical
signs, although only 8% lethality was reached. The disease spread rapidly to other farms over the island.
In the Havana province, 15 outbreaks affecting 23.201 animals, caused 10.547 deaths, and 5.256 animals
had to be slaughtered (Barrera et al., 2005; IMV, 2003).
The clinical signs of TGE are usually milder when TGEV is introduced into seropositive farms, or when
TGEV infects less susceptible animals, such as sows or finisher pigs in seronegative farms. Endemic TGE
is limited to seropositive herds and diarrhea can occur in pigs from 6 days old until 2 weeks after
weaning, and the mortality varies from 10-20% or even less. During this presentation TGE is difficult to
differentiate from rotavirosis or enteric colibacillosis (Saif and Sestak, 2006). TGEV infection therefore
occasionally goes undiagnosed. Risk for TGE was greater in herds with more than 50 breeding pigs than
in smaller ones.(p
-
Chapter 1
32
Rotavirus
Rotaviruses are RNA viruses of the Reoviridae family, which replicate mainly in the small intestine
and spread mainly via the fecal-oral route. The rotaviral genome consists of 11 segments of double
stranded RNA coding for six viral structural and six non-structural proteins (Matthijnssens et al., 2008).
The viral particles are composed of a capsid which contains three layers of viral proteins (VP) described
as the outer (glycoprotein VP7 and non-glycosilated protease-sensitive VP4), intermediate (VP6), and
inner (VP2) layers. VP1-3 are the core proteins (Yuan et al., 2006).
Rotavirus A is common in piggeries and is more prevalent in piglets from 1 to 3 weeks old and soon
after weaning (Atii et al., 1990). In pigs positive for rotavirus A, Halaihel et al. (2010) found that diarrhea
is more likely to occur in the ones younger than 8 weeks old. The prevalence of infection and disease is
favored by predisposing factors such as immunological quality and quantity of colostrum intake,
nutrition and the immune status of the sows, poor sanitary conditions in pens and around the piggeries,
and high population density (Steel and Torres-Medina, 1984; Atti et al., 1990; Zijlstra et al., 1999;
Barreiros et al., 2003). Additionally, the continuous exposure of pigs to rotaviruses is favored by their
resistance in the environment as they maintain infective for 32 months at 10°C in stool specimens
(Ramos et al., 2000).
Porcine rotaviruses are antigenically diverse. The two outer capsid proteins, VP7 (G genotype) and
VP4 (P genotype), independently elicit serotype-specific neutralizing immune responses that are
believed to play an important role in protection against recurrent infections (Santos and Hoshino, 2005).
Based on the differences in nucleic acid sequences of the outer capsid VP7 and VP4 encoding genes, 23
G and 31 P genotypes of rotavirus A have been described (Abe et al., 2009; Ursu et al., 2009). In pigs, 10
G types (1-6, 8-10 and 11) and 7 P types (5-8, 13, 9 and 23) of rotavirus A have been associated with
diarrhea (Martella et al., 2001; Barreiros et al., 2003). Group A rotavirus infection has been recognized
to occur in both enzootic and epizootic forms of swine diarrhea, resulting in serious economic losses in
the suckling and weaning piglet population of commercial piggeries (Martella et al., 2007; Kim et al.,
2010a).
The rotaviral replication in the villous epithelial cells cause malabsorption due to loss of absorptive
cells and villous atrophy, which is the more accepted mechanism of rotavirus induced diarrhea in pigs
(Greenberg and Estes, 2009). Rotavirus also induces an intestinal inflammatory response that may
contribute to a secretory-type diarrhea (Zijlstra et al., 1999), evokes intestinal fluid and electrolyte
secretion by activation of the nervous system in the intestinal wall as evidenced by the use of four
enteric nervous system inhibitor drugs (Lundgren et al., 2000). The rotaviral non-structural protein 4
-
Chapter 1
33
induces diarrhea in a similar way as E. coli STa by activating guanylate cyclase (Ball et al., 1996; Kavanagh
et al., 2010).
Colostrum-deprived piglets inoculated with rotavirus 24 hours after birth develop profuse diarrhea
with high mortality (63%). Interestingly, when these piglets were re-grouped with their colostrum-fed
litter mates, the later got infected and developed diarrhea with a mortality of only 8% and decreased
weight gains. Piglets recovered from rotavirosis can excrete the virus up to 21 days post-infection and
the severity of the infection is age dependent, and piglets inoculated at 5-14 days old developed
diarrhea but suffered a low mortality rate (Svensmark et al., 1989a). The highest rotavirus prevalence
occurs most frequently in litters from primiparous sows (Svensmark et al., 1989b). Sows are easily
infected with rotavirus by contact with an infected litter, but they do not show signs of diarrhea,
meaning that they are an important source of infection to their offspring (Svensmark et al., 1989a).
Moreover this contact leads to a better transfer of anti-rotavirus immunoglobulins through colostrum in
multiparous sows.
Field studies diverge concerning the pathogenicity of porcine rotaviruses. Rotavirus A was identified
in feces of 43 out of 96 (44.8%) piglets suffering from acute gastroenteritis, while none of 41 non-
diarrheic piglets were positive (P
-
Chapter 1
34
more sensitive and reliable method for G and P genotyping of group A rotavirus (Martella et al., 2001;
Barreiros et al., 2003). Chizhikov et al. (2002) recommended the oligonocleotide microarray
hybridization for the identification of the G genotypes of all rotavirus strains combining RT-PCR and
DNA-DNA hybridization. On swine stool specimens, Kang et al. (2007) suggested the use of
immunochromathographic assays that can separately and accurately detect porcine rotaviruses, TGEV
and PEDV.
In pigs, multiple rotavirus serogroups and serotypes have been detected worldwide (Table 8).
Table 7. Occurence of TGEV and PEDV detected by assays applied to serum and feces of pigs worldwide.
Groups & age Tested %
Assay Country/Reference TGEV PEDV
Serum
- 665 0 - VN U.S.A./Woods et al., 1990
Sows - 0.6 - Competitive ELISA Great Britain/Brown and Paton, 1991
- 229 89.9 - ELISA U.S.A./Phillips and Westerman, 1991
Breeding pigs 6000 1.27 - ELISA Spain/Cubero et al., 1993
Pigs in positive
farms - 5-60 - ELISA Spain/Cubero et al., 1993
Pigs in abattoirs 5.337 0 - ELISA South Africa/Williams et al., 1994
>7 months feral
swine 117 0 - IFAT U.S.A./Saliki et al., 1998
Wild boars 134 1 - IFAT Czech Republic/Sedlak et al., 2008
Wild boars 178 0 0 ELISA Slovenia/Vengust et al., 2006
- 263 12.5 - ELISA Japan/Miyazaki et al., 2010
Diarrheic feces
1-15 d. 749 59.2 - FAT and EM Canada/Morin et al., 1983
0-21 d. 1258 0.9 50.4 RT-PCR South Korea/Chae et al., 2000
1-7 d.
8-14 d.
15-21 d.
22-28 d.
36-42w
d.
33
50
19
16
31
0
4
10.5
0
51.6
EM Germany/Wieler et al., 2001
1-7 d. 100 6 - FAT and IHC U.S.A./Yaeger et al., 2002
1-14 d. 157 2.5 13.5 RT-PCR South Korea/Song et al., 2006
1-21 d. 153 0 0 RT-PCR Japan/Katsuda et al., 2006
22-35w
d. 116 0 0 RT-PCR Japan/Katsuda et al., 2006
2-28 d. 68 52 41.8 IC Czech Republic/Czanderlova et al.,
2010
w
, weaned; d., days old.
-
Chapter 1
35
Table 8. Worldwide occurrence (%) of Rotavirus in feces of diarrheic pigs during last decade.
Age group Tested % A B C Assay Country/Reference
1-60 d. DN
165 35.3 100 NT NT PAGE, ELISA, RT-PCR Brazil/Rácz et al., 2000
1-7 d. 100 42 100 NT NT ELISA U.S.A./Yaeger et al., 2002
1-7 d.
8-14 d.
15-21 d.
22-28 d.
36-42 d.
33
50
19
16
31
0
2
5.3
25
0
NT NT NT EM Germany/Wieler et al., 2001
< 7 d.
8-21 d.
> 21 d.
19
20
60
53
60
62
100 NT NT PAGE Brazil/Barreiros et al., 2003
1-7 d.
8-14 d.
15-21 d.
22-28 d.
29-35 d.
60
44
46
40
46
81.7
61.4
60.9
77.5
43.5
60-70* NI NI RT-PCR Japan/Katsuda et al., 2006
1-14 d. 157 10.8 100 NT NT RT-PCR South Korea/Song et al., 2006
Piglets 175 22.3 100 NT NT ELISA in fecal samples Thailand/Khamrin et al., 2007
1-3 m. 102
- 71.5
NT 31.3 RT-PCR
Italy/Martella et al., 2007 86** 81.3 25.5 EM, RT-PCR
< 45 d. DN
905 3.3 100 NT NT PAGE, ELISA, RT-PCR Argentina/Parra et al., 2008
< 3 w.
-
50
100 NT NT RT-PCR Slovenia/Steyer et al., 2008 3-10 w. 35.7
> 10 w. 46.2
Piglets 476 18.9 100 NT NT ELISA (fecal VP6) Czech Republic/Rodák et al.,
2009 21*** 76.2 NT 14.3 76.2 PCR
0-4 w. DN
4-8 w.
8-16 w.
16-24 w.
45
19
83
63
26.7
47
14.5
6.3
100 NT NT RT-PCR Spain/Halaihel et al., 2010
3-70 d. 475 38.3 100 NT NT RT-PCR, nested PCR South Korea/Kim et al., 2010a
1-7 d. 30 13.3 NI NI NI PAGE Brazil/Cruz et al., 2010
2-28 d. 98 30.6 100 NT NT IC Czech Republic/Czanderlova
et al., 2010
A, B and C, groups of Rotavirus (%); DN
, diarrheic and non-diarrheic pigs.*, from the positives; **, positives by EM;
***, samples EM+/ VP6 ELISA
-; NI, no information; NT, not tested; d., days old; w., weeks old, m., months old.
-
Chapter 1
36
Cryptosporidium
The contribution of Cryptosporidium to piglet diarrhea should not be ruled out as experimental
infections have revealed its potential pathogenicity (Suárez-Luengas et al., 2007). In two series of
experimental infections to compare the infectivity of the Cryptosporidium pig genotype I (C. suis) and C.
parvum in piglets, Enemark et al. (2003ab) reported that infection with C. suis caused no or very mild
clinical signs whereas C. parvum provoked diarrhea. The species or genotypes of Cryptosporidium can be
distinguished on the basis of the small subunit ribosomal RNA gene sequence (Xiao et al., 1999).
It has been suggested that the withdrawal of protective maternal antibodies and the stress of
weaning renders weaners particularly susceptible to infection (Hamnes et al., 2007). As referred by
Sanford (1987), Izumiyama et al. (2001) and Ryan et al. (2003), the higher positivity of pigs to
Cryptosporidium occurs in the post-weaning period and Chen and Huang (2007) reported that the
youngest pig excreting oocysts was 3 days old, and the oldest pig was 3.5 years old. Sows could maintain
the parasite life cycle in piggeries, even in the absence of piglets, because stress associated with
pregnancy and hormonal changes may increase susceptibility to infection (Zintl et al., 2007). Recently
Chen and Huang (2007) showed that Cryptosporidium isolated from pigs in eastern China belonged to
the C. parvum ‘mouse’ genotype indicating that rodents as sows play a role in swine cryptosporidiosis
epidemiology as reservoirs.
The specific mechanism by which C. parvum induces diarrhea has not been identified. Many
molecules like CSL, a 1.300-kDa conserved apical complex glycoprotein, are crucial for the adherence of
sporozoites and merozoites to the gut epithelium. Cryptosporidium gets internalized into the
enterocytes and locates in a membrane-bound compartment on the apical surface. Therefore, direct
damage of the brush border membrane, which provokes structural villous atrophy, and parasite derived
molecules cause malabsorption and a sequence of biochemical/immunological changes like increase of
secretogenic prostaglandins leading to diarrhea (Okhuysen and Chappell, 2002). Worldwide many
studies have associated this Apicomplexa with swine diarrhea. Chen and Huang (2007) found in China a
positive correlation between oocyst positivity and diarrhea, and Cryptosporidium prevalence was not
significantly different between pre and post-weaned piglets. Hamnes et al. (2007) reported in Norway a
significantly higher prevalence of diarrhea among the Cryptosporidium positive litters than among the
negative ones. In Serbia it was reported that all Cryptosporidium positive nursing piglets had diarrhea,
whereas in post-weaned piglets and adults Cryptosporidium infection was asymptomatic (Mišic et al.,
2003). In Mexico, Nevarez et al. (1997) implicated Cryptosporidium as infectious cause of enteritis in
three 10-week-old weaned piglets from a crowded herd with bad sanitation and subjected to massive
-
Chapter 1
37
treatment with several antibiotics. Conversely, in 20 out of 48 diarrheic pigs, C. parvum was not
determined to be the only cause of diarrhea, suggesting that it may act in concert with other agents (i.e.
E. coli, I. suis, Salmonella, adenovirus) to induce or exacerbate clinical disease and suggesting a strong
possibility of subclinical infection (Sanford, 1987). Similarly, Quilez et al. (1996), Ryan et al. (2003),
Guselle et al. (2003) and Vítovec et al. (2006) did not associate Cryptosporidium infection with swine
diarrhea. Enemark et al. (2003a) found that a mixed infection of rotavirus and Cryptosporidium caused a
dramatic aggravation of diarrhea. Vítovec et al. (2006), Maddox-Hyttel et al. (2006), and Johnson et al.
(2008a) reported that Cryptosporidium occurs more frequently in weaned pigs than in sucking piglets.
Table 9 shows the fecal occurrence of Cryptosporidium in pigs raised worldwide during the last
decade and several diagnostic techniques, which have been applied for its identification.
Isospora
The coccidian protozoa Isospora is one of the most common enteropathogens amongst suckling
piglets in intensive pig production units, and is an important enteropathogen to be surveyed and
controlled as the infection is associated with diarrhea (Harleman and Meyer, 1984; Vitovec and Koudela,
1990; Niestrath et al., 2002).
Piglets get infected by the ingestion of infectious oocysts from which the sporozoites emerge and
invade the epithelial cells of the small intestine. Later, the course of the life cycle phases merogony
(trophozoites, meronts, and merozoites), gametogony (macro and microgametocytes) and sporogony
(immature oocyst) contribute to the destruction of enterocytes and to the colonization of the intestinal
epithelium. Around four to five days after infection, piglets frequently have pasty to watery yellowish or
grayish diarrhea for 3-7 days, dehydration, and weight loss (Mundt et al., 2006). Clinical signs of
isosporosis mostly appear in suckling piglets in the first three weeks of life with high morbidity and low
mortality (Mundt et al., 2003).
Lindsay et al. (1992) and Otten et al. (1996) reported that isosporosis can occur in any farm,
independently of their size or management system. Niestrath et al. (2002) reported that the rearing
conditions and hygienic status on swine farms influence the prevalence of coccidian infections in piglets.
Traditionally, sows have been thought to be the main source of Isospora in a farm, but Karamon et al.
(2007) in Poland demonstrated no correlation between the excretion of oocysts by the sows and the
infection of piglets born from them. The farrowing pens contaminated with oocysts excreted by
previous litters is considered to be the main source of infection, showing the importance of good
sanitation (oocysts are resistant to most of disinfectants) and the use of self-cleaning floors in farrowing
-
Chapter 1
38
facilities for isosporosis prevention (Lindsay et al., 1989; Niestrath et al., 2002; Karamon et al., 2007). In
that context, Niestrath et al. (2002) commonly found I. suis in farms which employed straw bedding.
The general laboratory methods which included staining and or visualization of oocyst morphology
by light microscopy have been employed to identify Isospora but their sensitivity is negatively influenced
by the high fat content of piglets feces and because oocysts are not always excreted or are excreted
intermittently. Daugschies et al., 2001 reported that autofluorescence microscopy is more sensitive than
bright field microscopy for the detection of I. suis oocysts after flotation or in direct smears. Recently a
PCR–RFLP assay based on the rDNA ITS-1 region has been developed to identify Isospora (Samarasinghe
et al., 2007; Johnson et al., 2008b). Table 10 shows the assays mostly employed for the identification of
porcine Eimeriidae and their prevalence worldwide.
-
Ch
ap
ter
1
39
Ta
ble
9.
Wo
rld
wid
e r
ep
ort
ed
fe
cal
occ
urr
en
ce (
%)
of
Cry
pto
spo
rid
ium
in
pig
s d
uri
ng
la
st d
eca
de
.
Gro
up
s T
est
ed
%
C
. su
is
C.
Ge
n I
I C
. p
arv
um
C
. m
uri
s A
ssa
y
Co
un
try
/Re
fere
nce
1m
.
3 m
.
6 m
.
21
3
19
25
2
32
.3
42
.1
0.4
x x x
F
orm
ol-
eth
er
con
cen
tra
tio
n
Imm
un
ofl
uo
resc
en
t st
ain
ing
(A
)
Jap
an
/Izu
miy
am
a e
t a
l.,
20
01
Su
cke
rs
an
d
we
an
ers
28
7
1.4
x
C
arb
olf
uch
sin
sta
inin
g
Ge
rma
ny
/Wie
ler
et
al.
, 2
00
1
Ad
ult
s 5
89
1
0.5
x
A
cid
-fa
st s
tain
ing
S
ou
th K
ore
a/Y
u a
nd
Se
o,
20
04
< 5
w.
> 6
w.
So
ws
33
68
83
5
13
5
5.7
24
.1
0
x
A
nil
ine
-ca
rbo
l-m
eth
yl
vio
let
sta
inin
g
Sh
ea
the
r’s
flo
tati
on
, P
CR
Cze
ch R
ep
ub
lic/
Vít
ov
ec
et
al.
,
20
06
< 1
m.
1-4
w.
So
ws
48
8
50
4
24
5
6
71
4
Imm
un
ofl
uo
resc
en
t st
ain
ing
De
nm
ark
/Ma
dd
ox-
Hy
tte
l e
t
al.
, 2
00
6
1-7
d.
8-1
4 d
.
15
-21
d.
22
-28
d.
29
-35
d.
60
44
46
40
46
0
0
0
2.5
28
.3
Flo
tati
on
D
Jap
an
/Ka
tsu
da
et
al.
, 2
00
6
4-1
0 w
.
10
-24
w.
6-1
2 m
. g
ilts
4 y
. so
ws
3-4
y.
bo
ars
12
7
12
1
15
75
4
15
7.4
6.7
13
.3
0
x x x
x x
x
x
Au
ram
ine
ph
en
ol
sta
inin
g
Sh
ea
the
r su
ga
r fl
ota
tio
n,
PC
R
Ire
lan
d/Z
intl
et
al.
, 2
00
7
1-2
w m
.
2-6
m.
So
ws
75
42
25
30
.7
11
.9
16
x x
x x
Fo
rmo
l-e
the
r co
nce
ntr
ati
on
,
PC
R
Sp
ain